The goals of this proposal are to examine the events and genetic defects that underlie melanoma formation. Melanoma is the most aggressive and lethal form of skin cancer. Most benign nevi and melanomas have mutations activating the BRAF serine/threonine kinase, suggesting that BRAF activation is an important but insufficient step in tumorigenesis. p53 pathway alterations are also implicated in melanoma formation. While many functions have been ascribed to BRAF and p53, little is known about which if any of these functions is important in transforming normal melanocytes into melanoma cells. Furthermore, additional genetic defects that contribute this process have yet to be defined. Zebrafish strains that express human oncogenic BRAF and are mutant for p53 have been created. These strains reliably develop melanomas that are histopathologically similar to those in humans. Preliminary results indicate that binucleate, polyploid melanocytes in these mutants may give rise to tumors. In Aim 1, the events of melanoma formation will be examined. Transgenic approaches in zebrafish and long-term imaging of mammalian cells in culture will be used to determine the mechanism by which BRAF causes binuclearity. Cell sorting coupled with transplantation will be employed to determine whether binucleate cells can give rise to melanomas. In Aim 2, genetic defects that contribute to melanoma formation will be identified and characterized. In preliminary studies, the Kit receptor tyrosine kinase was identified as a suppressor of melanoma onset and invasion. Activities of Kit required for its melanoma suppression will be assessed. To identify additional melanoma genetic defects, array comparative genomic hybridization (aCGH) will be used to characterize recurrent chromosomal gains and losses in these tumors. aCGH data from human and zebrafish melanomas will be used to identify candidate oncogenes that will be tested in a novel, high-throughput assay for contribution to melanoma formation. These studies may identify diagnostic and prognostic indicators of disease as well as therapeutic targets for cancer treatment.